Chapter 1. General Introduction
1. Carbohydrates
1.1 Structural polysaccharides in plants
1.1.2 Hemicellulose
1.1.2.3 Arabinoxylans
Arabinoxylans are composed of a β-(1-4)-D-xylopyranosyl backbone, with one or more- L-arabinofuranosyl residues substituted at position 2 or 3 (Fig. 1.3). On the other hand, in many cases, hexoses and hexuronic acids are also present, but as minor constituents. The hemicellulose fraction is composed mainly of xylans that are linked to cellulose fibrils by hydrogen bonds. The xylose residues tend to be acetylated at positions O–2 and O–3, whereas other groups, such as ferulic acid, tend to be bound to positions O–2 or O–3 and lignin, forming cross-links that entrap the polymer (Cosgrove, 1999). A combination of enzymes is therefore required for the release of arabinoxylans from the cell matrix and for its hydrolysis (Saha, 2001; Saha, 2003). Arabinoxylans are polysaccharides found in the bran of grasses and cereals (wheat, rye, barley, oat, rice, corn and sorghum). They are the major hemicellulose component of flour and bran (dietary fibres). Since xylose and arabinose are both pentoses, arabinoxylans are usually classified as pentosans. Arabinoxylans are important in the baking industry. The arabinose units bind water and produce viscous compounds that affect the consistency of dough, the retention of gas bubbles from
fermentation in gluten-starch films, and the final texture of baked products (Saha, 2003). The structural features of hetero xylans are depicted below which shows a non- substituted xylopyranose (Fig. 1.3A) and substituted xylopyranose (Fig. 1.3B, Fig.
1.3C and Fig. 1.3D). D-xylopyranose can be substituted at O-2, O-3 and O-5 position with L-arabinose as shown in Fig. 1.3B, Fig. 1.3C and Fig. 1.3D, respectively. The schematic representation of xylan substituted with L-arabinose shows the structure of these xylans and enzymes acting on them (Fig. 1.4). The (1→4)-β-D-xylan endohydrolase acts and degrades only the main chain xylan (1→4)-β-D-xylopyranose) as shown in Fig. 1.4. The main chain D-xylopyranose ring can be singly or doubly substituted with L-arabinose and the α-L-arabinofuranosidases are capable of attacking both singly and doubly substituted xylans (Fig. 1.4). The non-reducing (1→4)-β-D-xylopyranose are acted upon by β-D-xylosidases (Fig. 1.4).
Fig. 1.3 Chemical structure of arabinoxylan. A) non-substituted D-xylopyranose, B) O-2 substituted D-xylopyranose, C) O-3 substituted D-xylopyranose and also the attachment point of ferulic acid to O-5 of L-arabinfuranose, D) O-2 and O-3 substituted D-xylopyranose with L-arabinofuranosyl residues (Cyran et
Red
Fig. 1.4 Schematic representation of a typical xylan component of hemicellulose and the respective enzymes acting on them (Numan and Bhosle, 2006; Saha, 2003). NR represent the non-reducing end; Red represents the reducing end and the arrows depict the enzymes acting at particular site viz. xylan endohydrolase, α-L-arabinofuranosidase and β-D-xylosidase.
1.1.2.4 Xyloglucans
Xyloglucan are composed of β-(1→4)-glucose units, substituted with (1→6)-D-xylose side chains (Fig. 1.5) (Fry, 1989; Brennan and Harris, 2010).
Xyloglucans are heavily branched β(1→4)-glucans (Fig. 1.5). Xyloglucan makes up 20-25% (dry weight) of the type I primary cell walls (all flowers except the grass family) of all dicots and monocot species except grasses (type II primary cell wall) and cereals, where they are found in very less extent (2-5%) (Fry, 1989).
Fig. 1.5 Schematic representation of xyloglcan structure showing β-(1→4)-glucose units substituted at (1→6) position with D-xylopyranose sugars (Brennan and Harris, 2010).
1.1.2.5 Arabinans
Arabinans, of cell-wall pectic-substances, comprise 1,5-α-linked-L- arabinofuranosyl main chain residues to which other L-arabinofuranosyl residues are 1,3-α and 1,2-α linked in either a comb-like or a ramified arrangement. The branched arabinan display a main chain of 1,5-α-linked-L-arabinofuranose substituted with L- arabinofuranosyl residues at 1,3 or 1,2 position as depicted in Fig. 1.6. The linear arabinan show a linear main chain of 1,5-α-linked-L-arabinofuranose (Fig. 1.7).
α-1,2- substituted L-arabinose
α-1,3- substituted L-arabinose
Fig. 1.6 Chemical structure of branched arabinan (http://www.brenda-enzymes.org, Technische Universitat Braunschweieg).
α-1,5-L-arabinfuranosyl linear chain
Fig. 1.7 Chemical structure of linear arabinan (http://www.brenda-enzymes.org, Technische Universitat Braunschweieg).
1.1.2.6 Galactans
Galactans are polymers of β-D-galactopyranose. They are sometimes included in the hemicellulose group, but a recent review by Scheller and Ulvskov (2010) suggested that they should not be included in the heterogeneous group of hemicelluloses. However, at present they are classified as a hemicellulose. Scheller and Ulvskov gave reason that since galactans appear to be part of pectin molecules, at least in the initial synthesis stage. The galactans are also found to be substituted with arabinosyl resides in arabinogalactans. These are of two types, Type I which is rich in 1,4 linkages and type II, contains terminal 1,3,5-linked arabinose or 3,5-linked arabinose substituted to main chain of galatan (e.g larchwood arabinogalatan).
Fig. 1.8 Arabinogalactan type II Structure from Larchwood (Heinze et al., 2005).
1.1.2.7 Mannans
Mannans are heteropolymers and contains β-(1→4)-D-mannopyranose backbone. The β-(1→4)-D-mannopyranose backbone is usually substituted with galactose or glucose.
1.1.2.7.1 Galactomannans
Galactomannan is composed of a β-(1→4)-D-mannopyranose backbone to which single D-galactosyl units are attached to C-6 of some of the D-mannosyl residues as shown in Fig. 1.9 (McCleary and Matheson, 1983; Cui et al., 2009). The extent of galactopyranose substitution invariably affects the solubility, viscosity and interaction with other polysaccharides (Cui 2009).
galactopyranose
β-(1→→→→4)-D-mannopyranose
C-6 of D-mannaopyranose
main chain
β-(1→→→→4)-linkage
Fig. 1.9 Chemical structure of galactomannan (Cui et al., 2009).
1.1.2.7.2 Glucomannan
Glucomannans are the hetero-polymers of β-(1→4)-D-mannopyranose backbone and β-1,3 substituted β-D-glucopyranose (Fig. 1.10). They are generally found in the secondary walls of softwood (Maeda et al., 1980).
β-1,3-linked
β-1,4-linked
β-D-glucopyranose
β(1→→→→4)-D-mannopyranose
Fig. 1.10 Chemical structure of glucomannan (Maeda et al., 1980).
1.1.3 Lignin
The main building blocks of lignin are the hydroxycinnamyl alcohols (or monolignols) coniferyl alcohol and sinapyl alcohol, with typically minor amounts of p-coumaryl alcohol (Fig. 1.11). Lignins are large group of aromatic polymers that result from the oxidative combinatorial coupling of 4-hydroxyphenylpropanoids as shown in Fig. 1.11 (Boerjan et al., 2003; Ralph et al., 2004). Lignin is found in all
vascular plants, mostly between the cells and also within the cells and in the cell walls.It usually occurs as complex structure bound to the hemicelluloses in wood.
Fig. 1.11 The main biosynthetic pathway for synthesis of the monolignols p- coumaryl, coniferyl, and sinapyl alcohol (Boerjan et al., 2003).
Phenylalanine ammonia-lyase (PAL); Cinnamate-4-hydrolyase (C4H); 4- coumarate:CoA ligase (4CL); p-coumarate-3-hydrolyase (HCT), p- hydroxycinnamoyl-CoA:Quinate/Shikmate-p-hydroxycinnamoyltranferase (CCoAOMT), Caffeoyl-CoA-O-methyltransfersae (CCR), Cinnamoyl CoA reductase ferrulate-5-hydroxylyase (F5H), Caffeic acid-O- methyltransferase (COMT); Cinnamoyl alcohol dehydrogenase (CAD) (Adapted from Borjan et al., 2003 and Ralph et al., 2004).
The units resulting from the hydroxycinnamyl alcohols, when incorporated into the lignin polymer, are called guaiacyl (G), syringyl (S), and p-hydroxyphenyl (H) units (Figs. 1.11 and Fig 1.12). The β-O-4 linked aryl ethers are found most abundantly in the syringyl lignin of poplar (Fig.1.12). Apart from this, guaicyl (G), sinapyl p-hydroxybenzoate-derived, phenyl coumaran, biphenyl ether, resinol units were also present as components of the poplar lignin (Fig. 1.12).
S syringyl G Guaicyl
SP sinapyl p-hydroxybenzoate-derived β-O-4, β-aryl ether
β-5, phenyl coumaran β-β, resinol
4-O-5, biphenylether Cinnamyl alcohol endgroup Phenolic endgroup
biphenylether
Cinnamoyl alcohol end
group β-O-4
SP
Phenyl coumaran
Fig. 1.12 A schematic representation of a lignin polymer from poplar, as predicted from NMR-based lignin analysis. The poplar lignin polymer of around 22 units shows high S (syringyl) lignin with a prevalence of β-aryl ether linkages. Sinapyl p-hydroxybenzoate derived, phenyl coumaran, resinol, biphenyl units were also present in the poplar lignin polymer (Adapted from Boerjan et al., 2003 and Stewart et al., 2009).
1.1.4 Pectin
Pectins are a family of complex polysaccharides that contain 1,4-linked α-D- galactosyluronic residues. Three pectic polysaccharides, homogalacturonan, rhamnogalacturonan-I and substituted galacturonans have been isolated from primary plant cell walls (Ridley et al., 2001). Homogalacturonan is a linear chain of 1,4-linked α-D-galactosyluronic residues, in which some of the carboxyl groups are methyl esterified (Fig. 1.13). Homogalacturonan may also be O-acetylated at the C-2 and C-3
backbone of the repeating disaccharide [→4)-α-D-GalpA-(1→2)-α-L-Rhap-(1→] as shown in Fig. 1.14 (Ridley et al., 2001). The minor component of the pectin backbone is rhamnogalacturonan-II. This is not structurally related to rhamnogalacturonan-I since its backbone is composed of 1,4-linked α-D-galactosyluronic units (dimers) like homogalacturonan (Fig. 1.15). Rhamnogalacturonan-II is present in primary walls predominantly as a dimer of 1,4-linked α-D-galactosyluronate that is cross-linked by a 1:2 borate-diol ester as shown in Fig. 1.15 (Kobayashi et al., 1996; Ishii et al., 1999).
This cross-link is formed between OH-2 and OH-3 of the apifuranosyl residues (βApif) in each monomeric rhamnogalactoronan-II subunit (Fig. 1.15).
Rhamnogalactoronan-II is of interest as it occurs in relatively high amounts in wine and other fruit juices and it has been demonstrated that it binds heavy metals and has immunomodulating activities (Ridley et al., 2001).
Fig. 1.13 Chemical structure of homogalacturonan showing 1,4-linked α-D- galactopyranosyluronic acid residues (GalpA). The carboxyl groups of the GalpA residues are often methyl-esterified. Some of the hydroxyl groups may be O-acetylated (Ridley ., 2001).
Fig. 1.14 A schematic presentation showing the major structural features of rhamnogalacturonan I. The backbone is composed of the disaccharide repeating unit [1→4- α-D-galactopyranoseA-(→2)-α-L-rhamnopyranose- (1→]. Branched and linear oligosaccharides composed predominantly of α-L-arabinofuranose and β- D -galactopyranose residues are linked to C4 of some of the rhamnopyranose residues. Some of the rhamnopyranose residue may also be O-acetylated at C2 and/or C3 (Ridley et al., 2001).
A
Fig. 1.15 The primary structure of rhamnogalacturonan II showing backbone of 1→4-linked α-D-GalpA dimers. Four structurally different oligosaccharide side chains (A, B, C and D) are linked to the rhamnogalacturonan II backbone. The two disaccharides, α-L-Rhap- (1→5)-Kdo (3-deoxy-D-manno-octulopyranosylonic acid) and β-L- Araf- (1→5)-Dha, which are attached to position 3 of two of the 4-linked GalpA residues, have been omitted. The abbreviations used for different units are 3-C-carboxy-5-deoxy-l-xylofuranosyl (αAcef), arabinofuranosyl (Araf), arabinopyranosyl (Arap), fucopyranosyl (Fucp), galactopyranosyl(Galp), galactopyranosyluronic acid (GalpA), glucopyranosyluronic acid (GlcpA), 2-O-methylfucopyranosyl (2-O-Me- Fuc ), 2- -methylxylopyranosyl (2- MeXyl ) (Iishi ., 1999).
1.2 Carbohydrate-active enzymes
Carbohydrates are dynamic molecules that are constantly synthesized and broken down. There are varieties of enzymes involved in the synthesis as well as breakdown of carbohydrates. The glycosyltransferases (GTs) are mainly involved in the formation of the glycosidic bond or biosynthesis of carbohydrates. The polysaccharide lyases (PLs), carbohydrate esterase (CEs) and glycoside hydrolases (GHs) are concerned with the breakdown of polysaccharides. In summary, the carbohydrate-active enzymes are grouped into 262 families based on amino acid sequence similarity and are listed in the continually updated carbohydrate-active enzyme (CAZy) database (www.cazy.org). Out of 262 carbohydrate-active enzymes, about 130 families belong to GHs (Cantarel et al., 2009). These enzymes belonging to 130 GH families have been reported from nearly 1884 species of bacteria, 130 species of archaea and 65 species of eukaryote (www.cazy.org). A close inspection of the genomes listed within the database reveals the fact that 1-3% of the genome of most organisms is devoted to encoding glycosyltransferases (GTs) and glycoside hydrolases (GHs) (www.cazy.org). The information available at CAZy database provide a wealth of gene sequences (many yet to be characterized) to study the structure and function of carbohydrate-active enzymes (Cantarel et al., 2009).
1.2.1 Glycosyltransferases
Glycosyltransferases (GTs) are enzymes that catalyse the transfer of any sugar moieties from activated donor molecules to specific acceptor molecules, forming glycosidic bonds (Sinnot, 1990). These enzymes utilize 'activated' sugar phosphates as glycosyl donors and catalyze glycosyl group transfer to a nucleophilic group, usually an alcohol (Campbell et al., 1997). The product of glycosyl transfer
oligosaccharide or polysaccharide (Lairson et al., 2008). As of now, nearly 94 families of GTs have been recognized and are listed in the CAZy database (http://www.cazy.org/GlycosylTransferases.html). There are almost over 8000 gene sequences in GenBank and the crystal structure of 36 GTs has been solved to date (http://www.cazy.org/GlycosylTransferases.html). Many GTs have been reported from a wide range of bacterial population viz. Acidophilium, Actinoplanes, Bacillus, Clostridium, Gloeobacter, Lactobacillus, etc (Coutinho et al., 2008).
1.2.2 Polysaccharide lyases
Polysaccharide lyases (PLs) are group of enzymes that break the uronic acid- containing polysaccharide chains following a β-elimination mechanism to liberate an unsaturated hexenuronic acid residue and a new reducing end (http://www.cazy.org/Polysaccharide-Lyases.html). The CAZy database contains 22 families of these enzymes classified, based on amino acid sequence similarities, which also reflect their structural features (Lombard et al., 2010). As of now, more than a thousand gene sequences of PLs have been deposited in the GenBank and out of them 546 sequences are from bacteria. Nearly 98 PLs have been characterized to date and the crystal structures of only 12 PLs have been solved (http://www.cazy.org/Polysaccharide-Lyases.html).
1.2.3Carbohydrate esterase
The carbohydrate esterase (CEs) catalyzes the de-O or de-N-acylation of substituted saccharides. Two types of substrates have been considered for carbohydrate esterases: i) those in which the sugar plays the role of the "acid", such as pectin methyl esters for 4-O-methyl-glucuronoyl methylesterase from Schizophyllum commune (Li et al., 2007) and ii) those in which the sugar behaves as
the alcohol, such as acetylated xylan for acetyl xylan esterase (family 1 and 2 CEs) from Clostridium thermocellum ATCC 27405 (Montanier et al., 2009).
1.2.4 Glycoside hydrolase
Glycoside hydrolases (GHs) are enzymes that catalyze the hydrolysis of the glycosidic linkage of glycosides, leading to the formation of a sugar hemiacetal or hemiketal and the corresponding free aglycone. Glycoside hydrolases are also referred to as glycosidases, and sometimes also as glycosyl hydrolases. Glycoside hydrolases can catalyze the hydrolysis of O-, N- and S-linked glycosides. The glycoside hydrolases are a group of enzymes that exists in most living organism (Cantarel et al., 2009). They hydrolyze the glycosidic linkages between two or more carbohydrates or between a carbohydrate and a non-carbohydrate moiety (http://www.cazy.org/Glycoside-Hydrolases.html). The carbohydrate-active enzymes database (CAZy) provides a continuous updated list of the glycoside hydrolase families. They are classified into different families based on homology of their primary sequence (Henrissat, 1991). As of now, the GHs are grouped into 130 families based on amino acid sequence with more than 30,000 entries in the CAZy database (www.cazy.org). GHs are also grouped into 14 clans based on the fold of proteins as it was found to be better conserved than their amino acid sequence (Cantarel et al., 2009). Even the closely related catalytic GHs are very often found to differ in substrate specificity (http://www.cazy.org/), while enzymes with very different enzyme activities are found among glycoside hydrolase homologs (Coutinho et al., 2003). Small changes in the primary structure of glycoside hydrolases are able to change their substrate specificity (Andrews et al., 2000). Unlike other CAZymes (GTs, CLs, and CEs), the structural data on GHs has clearly displayed several different folds, such as (α/α) , β-helix, β-propeller, β-jelly roll and the (α/β) TIM
barrel motif. The (α/β)8 TIM barrel is found in the majority of GHs listed in CAZy database (Cantarel et al., 2009).
GHs are important plant cell wall degrading enzymes and thus cell wall degradation by microbial enzymes is pivotal to many biological and industrial processes (Gilbert et al., 2010). The polysaccharides of plant cell walls are relatively recalcitrant to enzymatic degradation and due to this the microbes, with time, have evolved and developed a complex enzymatic systems in order to counter this problem.
For example, Clostridium thermocellum and Clostridium cellulolyticum secrete a mega-dalton multi-modular enzyme complex called the “cellulosome” (Bayer et al., 2006; Fontes and Gilbert, 2010). The cellulosome is a macromolecular complex, whose components interact in a synergistic manner to catalyze the efficient degradation of cellulose. The cellulosome complex is composed of numerous kinds of cellulases and related enzyme subunits, which are assembled into the complex by virtue of a unique type of scaffolding subunit (Bayer et al., 1998; Bayer et al., 2004).
1.2.4.1 Glycoside hydrolases and their modular nature
A module is defined as a contiguous amino acid sequence within a larger sequence that folds independently and has an individual function but together increase the overall efficiency of the enzyme (Doi and Kosugi, 2004). The glycoside hydrolases are often found to exhibit a modular architecture comprising a catalytic module fused or attached with one or more ancillary modules via linker peptides. The ancillary modules are usually the carbohydrate binding modules (CBMs). The first indication that these enzymes contained distinct independent functioning modules was reported by Davies and Henrissat (1995, 2002) and later many researchers reported the same (Bayer et al., 2004). Fig. 1.16 shows typical example of a modular family 33
glycoside hydrolase (GH33) from Micromonospora viridifaciens displaying a catalytic GH33 module which is attached to a non catalytic family 22 carbohydrate binding module (CBM22) via a linker. The catalytic GH33 from Micromonospora viridifaciens was found to exhibit sialidase activity (Gaskell et al., 1995).
Non-catalytic CBM32 Linker region Catalytic GH33
Fig. 1.16 The 3-dimensional structure (PDB Code 1EUT) of full length family 33 glycoside hydrolase (MvGH33, sialidase) from Micromonospora viridifaciens as reported by Gaskell et al. (1995). The catalytic GH33 is shown in green; linker in yellow and carbohydrate-binding module (CBM32) is shown in red.
1.2.4.2 Cellulosome structure
The cellulosome is a macromolecular complex, whose components interact in a synergistic manner to catalyze the efficient degradation of cellulose (Bayer et al., 2007). The cellulosome complex comprises numerous kinds of cellulases and related enzyme subunits, which are assembled into the complex by virtue of a unique type of scaffolding subunit known as scaffoldin (Bayer et al., 2004). The cellulosomal
enzymes from Clostridium thermocellum range in molecular size from about 40 to 180 kDa (Fontes and Gilbert, 2010). The realization about the multi-enzyme complex (cellulosome) occurred gradually with discoveries of different catalytic and binding domains, dockerins and scaffoldins, first in clostridial species and then subsequently in other bacteria (Doi et al., 1994; Belaich et al., 1997; Doi and Kosugi, 2004). Each of the cellulosomal subunits consists of a multiple set of modules, two classes of which (dockerin domains on the enzymes and cohesin domains on scaffoldin) govern the incorporation of the enzymatic subunits into the cellulosome complex (Bayer et al., 1998). The cellulosomal enzymes are usually members of the glycosyl hydrolase families of enzymes, which hydrolyze oligosaccharides and polysaccharides (Henrissat, 1991; Fontes and Gilbert, 2010).
1.2.4.3 Glycoside hydrolases and their activity
The glycosidic bond linking two carbohydrates together or a carbohydrate to another non-sugar (aglycone) compound is generally a very stable bond with an estimated half life of 5 million years (Wolfenden et al. 1998). The GHs act to hydrolyze these glycosidic bonds and thereby increase bond cleavage efficiency up to
~ 1017 fold (Wolfenden et al. 1998). The substrates degraded by GHs include plant cell wall polysaccharides such as cellulose, hemicellulose and lignin.
1.2.4.4 Mechanism of action of Glycoside hydrolase
Glycoside hydrolases usually hydrolyze glycosidic bonds by adding water across the glycosidic bond. Asp or Glu are the most commonly found catalytically active residues, although a few exceptions do exist (Davies et al., 2002; Rajan et al., 2003). There are two fundamental mechanisms of action by which these enzymes
hydrolyze the substrates (www.cazy.org). These include i) Retaining mechanism and ii)Inverting mechanism and are explained in the sub sections 1.2.4.4.1 and 1.2.4.4.2.
1.2.4.4.1 Retaining mechanism
Glycoside hydrolase acting by retaining mechanism generally have a nucleophile and catalytic acid/base residues approximately 6Å apart (Zechel and Withers, 2000). This reaction proceeds via a double displacement mechanism (Fig 1.17A). In the first step, the nucleophilic attack occurs at the anomeric centre with immediate protonation of the glycosidic oxygen by the general acid (Fig 1.17A). This occurs through a transition state that has oxocarbenium ion character and results in the formation of a glycosyl-enzyme intermediate and results in retention of stereochemistry at the anomeric carbon as shown in Fig. 1.17A (Zechel and Withers, 2000). Hydrolysis of the intermediate occurs and simultaneously, the general base deprotonates an incoming water molecule, which further attacks the anomeric centre causing hydrolysis of the glycosyl-enzyme intermediate (Fig 1.17A).
1.2.4.4.2 Inverting mechanism
Inverting glycoside hydrolases require a larger distance between general base and general acid residues in order to accommodate the direct attack of a water molecule and the substrate (Zechel and Withers, 2000). This reaction proceeds via a single displacement mechanism (Fig. 1.17B). Water is deprotonated by the general base and attacks the anomeric centre while the general acid concomitantly protonates the leaving group (Fig. 1.17B). The inverting mechanism proceeds via an oxacarbenium ion-like transition state and results in overall inversion of stereochemistry at the anomeric carbon (Zechel and Withers, 2000).